The long-term objective of this laboratory is to define the role of the red blood cell (RBC) as a participant in the control of vascular resistance. Previously, we reported that, in the pulmonary circulation of the rabbit, RBCs of either rabbits or healthy humans were a required component of the perfusate in order to demonstrate flow-induced endogenous nitric oxide (NO) synthesis. This property of RBCs was found to reside in the ability of these cells to release ATP in response to a physiological stimulus (deformation). In this construct, as the RBC is deformed by the velocity of blood flow through a vessel and/or by reductions in vascular caliber, it releases ATP which stimulates endothelial NO synthesis resulting in an increase in vascular caliber. In addition to deformation, exposure of RBCs to reduced oxygen tension stimulates ATP release. It was proposed that, via this property, RBCs participate in the regulation of blood flow within a tissue such that blood flow is matched to metabolic need. ATP release from the RBC requires a specific release mechanism. We have demonstrated that a signal-transduction pathway that includes the cystic fibrosis transmembrane conductance regulator (CFTR), protein kinase A, adenylyl cyclase/cAMP and the heterotrimeric G proteins Gs and Gi is responsible for deformation-induced ATP release from rabbit and human RBCs. The overall goal of our work remains the definition of the contribution of the RBC to the local control of vascular caliber. The aims of the current proposal are to demonstrate that 1) mechanical deformation and exposure to reduced 02 stimulate a common signal-transduction pathway for ATP release, 2) decreases in cell deformability result in failure to release ATP in response to these stimuli, 3) RBCs of rabbits and humans posses adenylyl cyclase isoforms and G protein beta subunits that can interact resulting in cAMP generation, and 4) pharmacological agents and endogenous autacoids that activate heterotrimeric G proteins or stabilize RBC cAMP stimulate ATP release from RBCs of rabbit and healthy humans.